(1) Field of the Invention
The present invention relates to methods of operating a microscope, in general, and to a method of operating a laser scanning microscope having at least two independently controlled scanning systems.
(2) Description of Related Art
Confocal laser microscopy is, among other things, the tool for the defined control of micro objects. Versatile methods of examining and influencing microscopic objects were recommended on this basis—e.g., Denk in U.S. Pat. No. 5,034,613, TPA, Liu in U.S. Pat. No. 6,159,749, Tweezer or Karl Otto Greulich in “Micromanipulation by Light in Biology and Medicine” 1999. A combination of a point-scanning or line-scanning imaging system and a “manipulator” system has evoked increasing interest in the specialized world.
Interest in observing and analyzing fast microscopic processes has created new devices and processes (e.g., line scanner LSM 5 LIVE), whose combination with the manipulation methods mentioned above leads to new insights. In this context, the simultaneous microscopic observation of a light induced, locally resolved sample manipulation with the help of a suitable imaging system occupies the foreground (U.S. Pat. No. 6,094,300 and DE 102 004 03 4987 A1). Modern microscopes therefore try to offer the maximum possible number of flexible and optically equivalent coupling and decoupling positions (DE 102 004 01 6433 A1).
The simultaneous availability of at least two coupling positions for independent scanning systems is very important in this context for avoiding limitations in time resolution due to slow mechanical control processes. In addition to the tube interface, there are other coupling positions on the sides of the microscope stands (preferably in an extended infinite space between the microscope objective and tube lens; “side ports”) as well as on the rear side of the stand (typically optically modified reflected or transmitted light axes with suitable tube lens; “rear ports”) as well as the bottom side (“base port”). In principle, arrangements with a common beam direction (either reflected light or transmitted light) or the opposite beam direction (reflected light and transmitted light) are possible. Apart from the practical background, the technical instrument-based view of the common beam direction is often preferred.
At least one element must be used in this case that combines the beam paths of the two instruments in the space between the scanners of the simultaneously operated scanning systems and the objective. According to the prior art, one can think of the most varied of beam-combining elements such as for instance, optical-mechanical components like suitably coated beam-combining, flat plates and beam-combining wedges, beam-combining cubes and a polarization splitter. Further, beam-combining acousto-optical modulators and deflectors are also conceivable.
The mechanical requirements related to the precision of location and angle of this beam-combining element are very high. A faulty installation angle α causes tilting of a beam inclination by 2α in reflection. For example, if the beam-combining element is in the infinite space between a tube lens of focal length fTL=164 mm and an objective of the nominal foreground M=fTL/fObj=40× then this leads to an angular deviation of 2α=1′ (position deviation of the beam combiner 0.5′) to a deviation Δ=(fTL/M)*tan 2α=1.2 μm of both scan fields in the object plane. In a field of view 18 (diagonal) this already corresponds to a deviation of approximately 0.4% of the lateral length of the scan field. In the usual image formats of 512×512 or 1024×1024, this corresponds to a deviation of 2-4 image pixels. In addition to the demanding mechanical requirements related to the mechanical positioning of the beam-combining element, there are similarly demanding tolerance specifications related to the mechanical interfaces of the imaging or manipulation scanning module (inclination errors and lateral shifting of interface, intermediate image position in axial direction, and rotation). Further, thermal influences (heating of the microscope system, and fluctuations in the environmental temperature) as well as undefined statistical effects, impose a condition that occurs especially in case of extremely precise measurements, the coverage of the scan fields in the manipulating and imaging systems must be adjusted repeatedly.